PROJECT SUMMARY The goal of this proposal is to understand how Musashi-2 (MSI2) affects resistance to treatment of non-small cell lung cancers (NSCLC) to inhibitors of the epidermal growth factor receptor (EGFR) receptor tyrosine kinases. This project arose from our earlier comparison of cell line panels derived from non-metastatic versus highly metastatic tumors of the KrasLA1/+; P53R172H?G/+ (KP) mouse model for human NSCLC, which for the first time identified upregulation of Musashi-2 (MSI2) as one of the most consistent features of metastatic cells. From analysis of two independent sets of primary human tissue specimens, we determined that MSI2 expression is significantly elevated during tumor progression, and predicts poor outcome. Initial functional analysis defined a mechanism by which MSI2 upregulates the transforming growth factor beta receptor (TGFBRI) and SMAD3, and downregulates claudins, to promote invasion and a mixed epithelial/mesenchymal phenotype. While revealing MSI2 mechanism of action, this did not have direct relevance to clinical practice. Using reverse-phase protein array (RPPA) screening of NSCLC Msi2-depleted cells, coupled with subsequent validation, we determined that MSI2 positively regulated EGFR and negatively regulated HER2 in NSCLC. Interestingly, MSI2 depletion in EGFR mutant, but not in EGFR wild type NSCLC cell line resulted in a dramatic inhibition of cell proliferation. EGFR mutations are found in ~15% NSCLC, and are the most common targetable mutations in this disease; further, the EGFR inhibitors erlotinib and afatinib are highly efficacious FDA-approved drugs in EGFR-mutant NSCLC, and also show some activity in a subset of EGFR-wild type NSCLC patients. We hypothesize that MSI2 regulation of the expression of EGFR and HER2 influences EGFR/HER2-dependent signaling cascades and biases the resistance profile of NSCLC to drugs targeting EGFR and/or HER2. In this proposal, we will complement investigations of MSI2 regulation of EGFR and HER2 signaling with preclinical studies addressing the importance of targeting MSI2 together with EGFR and HER2 signaling pathways. We will use clinically relevant EGFR wild type and EGFR mutant NSCLC cell line, xenograft, and transgenic models, and we will test FDA-approved EGFR inhibitor erlotinib, alone and in combination with novel first-in-class Musashi inhibitors we have developed. In Aim 1, we will evaluate MSI2 as a regulator of response to EGFR inhibitors, in vitro and in vivo, and evaluate the impact of genetic ablation of MSI2 in a mouse model. In Aim 2, we will evaluate the effect of combining novel small molecule MSI2 inhibitors with EGFR inhibitors.